This invention generally relates to the synthesis of paclitaxel and paclitaxel analogs from precursor compounds. More particularly, though, this invention concerns the synthesis of paclitaxel and analogs thereof through the step of esterifying C-7, C-10 di-CBZ 10-deacetylbaccatin III with a suitably protected 3-phenylisoserine side chain, followed by subsequent deprotections and acylations. The present invention also relates to methods of acylating C-2xe2x80x2-O-protected-10-hydroxy-paclitaxel and its analogs selectively at the C-10 hydroxy position over the C-7 hydroxy position. The present invention further relates to C-10 metal alkoxide intermediate compounds useful in producing paclitaxel and paclitaxel analogs.
The chemical compound referred to in the literature as taxdl, and more recently xe2x80x9cpaclitaxelxe2x80x9d, has received increasing attention in the scientific and medical community due to its demonstration of anti-tumor activity. Paclitaxel has been approved for the chemotherapeutic treatment of several different varieties of tumors, and the clinical trials indicate that paclitaxel promises a broad range of potent anti-leukemic and tumor-inhibiting activity. As is known, paclitaxel is a naturally occurring taxane diterpenoid having the formula and numbering system as follows: 
While the paclitaxel molecule is found in several species of yew (genus Taxus, family Taxaceae), the concentration of this compound is very low. Moreover, these evergreens are slow-growing. Thus, a danger exists that the increasing use of paclitaxel as an effective anti-cancer agent will deplete natural resources in the form of the yew trees. Indeed, while the bark of the yew trees typically exhibit the highest concentration of paclitaxel, the production of 1 kilogram of paclitaxel requires approximately 16,000 pounds of bark. Thus, the long term prospects for the availability of paclitaxel through isolation is discouraging.
The paclitaxel compound, of course, is built upon the baccatin III backbone, and there are a variety of other taxane compounds, such as baccatin III, cephalomannine, 10-deacetylbaccatin III, etc., some which are more readily extracted in higher yields from the yew trees. Indeed, a relatively high concentration of 10-deacetylbaccatin III can be extracted from the leaves of the yew as a renewable resource. Typically, however, these other taxane compounds present in the yew tree do not exhibit the degree of anti-tumor activity shown by the paclitaxel compound.
Since the paclitaxel compound appears so promising as a chemotherapeutic agent, organic chemists have spent substantial time and resources in attempting to synthesize the paclitaxel molecule. A more promising route to the creation of significant quantities of the paclitaxel compound has been proposed for the semi-synthesis of paclitaxel by the attachment of the A-ring side chain to the C-13 position of the naturally occurring baccatin III backbone derived from the various taxanes present in the yew. See, Denis et al, a xe2x80x9cHighly Efficient, Practical Approach to Natural Taxolxe2x80x9d, Journal of the American Chemical Society, page 5917 (1988). In this article, the partial synthesis of paclitaxel from 10-deacetylbaccatin III is described.
The most straightforward implementation of partial synthesis of paclitaxel requires convenient access to a chiral, non-racemic side chain and derivatives, an abundant natural source of baccatin III or closely related diterpenoid substances, and an effective means of joining the two. Of particular interest then is the condensation of baccatin III or 10-deacetylbaccatin III with the paclitaxel A-ring side chain. However, the esterification of these two units is difficult because of the hindered C-13 hydroxyl of baccatin III located within the concave region of the hemispherical taxane skeleton. For example, Greene and Gueritte-Voegelein reported only a 50% conversion after 100 hours in one partial synthesis of paclitaxel. J. Am. Chem. Soc., 1988, 110,5917.
In U.S. Pat. No. 4,929,011 issued May 8, 1990 to Denis et al entitled xe2x80x9cProcess for Preparing Taxolxe2x80x9d, the semi-synthesis of paclitaxel from the condensation of a (2R,3S) side chain acid of the general formula: 
wherein P1 is a hydroxy protecting group with a taxane derivative of the general formula of: 
wherein P2 is a hydroxy protecting group is described wherein the condensation product is subsequently processed to remove the P1 and P2 protecting groups. In Denis et al, the (2R,3S) 3-phenylisoserine derivative, with the exception of the P1 protecting group, is the A-ring side chain for the paclitaxel molecule. The P2 protecting group on the baccatin III backbone is protected by, for example, a trimethylsilyl or a trialkylsilyl radical.
An alternative semi-synthesis of paclitaxel is described in U.S. Pat. No. 5,770,745 to Swindell et al. That patent discloses semi-synthesis of paclitaxel from a baccatin III backbone by the condensation with a side chain having the general formula: 
wherein R1 is alkyl, olefinic or aromatic or PhCH2 and P1 is a hydroxyl protecting group.
The side chain in Swindell et al is distinct from the side chain attachment used in Denis et al, above in that the nitrogen is protected as a carbamate. Preferably, the A-ring side chain is benzyloxycarbonyl (CBZ) protected. After esterification, the CBZ protecting group is removed and replaced by PhCO to lead to paclitaxel. This process generated higher yields than that described in Denis et al. In Swindell et al, the preferred masking groups were selected to be trichloroethoxymethyl or trichloroethoxycarbonyl. Benzyloxymethyl (BOM) was, however, disclosed as a possible side chain protecting group, but, according to the processes described therein, the BOM protecting group could not be removed from the more encumbered C-2xe2x80x2 hydroxyl in the attached 3-phenylisoserine side chain. The use of the BOM protected side chain was not extensively investigated, for that reason.
U.S. Pat. No. 5,675,025 issued Oct. 7, 1997 to Sisti et al describes methodology for successfully using the C-2xe2x80x2OBOM side chain in paclitaxel synthesis. More particularly, the ""025 Patent teaches a method to remove the C-2xe2x80x2OBOM group in C-2xe2x80x2OBOM paclitaxel to produce paclitaxel.
U.S. Pat. No. 5,684,175 to Sisti et al and WO 96/40666 each describe the production of paclitaxel which includes esterfying a suitably protected side chain with a C-7 TES protected baccatin III. Notably, the C-10 acetate is present prior to the attachment of the C-13 side chain.
U.S. Pat. No. 4,924,012, issued May 8, 1990 to Colin et al discloses a process for preparing derivatives of baccatin III and of 10-deacetylbaccatin III, by condensation of an acid with a derivative of a baccatn III or of 10-deacetylbaccauin III, with the subsequent removal of protecting groups by hydrogen. Several syntheses of TAXOTERE(copyright) (Registered to Rhone-Poulenc Sante) and related compounds have been reported in the Journal of Organic Chemistry: 1986, 51, 46; 1990, 55, 1957; 1991, 56, 1681; 1991, 56, 6939; 1992, 57, 4320; 1992, 57, 6387; and 993, 58, 255; also, U.S. Pat. No. 5,015,744 issued May 14, 1991 to Holton describes such a synthesis.
European Patent No. 0522958A1 appears to relate to the preparation of various derivatives of baccatin III and 10-deacetybaccatin III, and particularly ones having C-7 and/or C-10 protecting groups. In particular, that reference appears to teach the esterification of an appropriate paclitaxel or docetaxel side chain with a suitably protected baccatin III or 10-deacetylbaccatin III backbone.
WO 98/13360 teaches a method for paclitaxel synthesis that includes esterfying C7-CBZ baccatin III with C-3xe2x80x2 N-CBZ -C2xe2x80x2-O-protected (2R, 3S)-3-phenyl isoserine, and thereafter performing varous deprotections and acylations to obtain paclitaxel.
WO 98/02427 teaches a method of converting 10-deacetylbaccatin III to baccatin III by acylating 10-deacetylbaccatin III selectively at the C-10 position over the C-7 hydroxy position thereof. The selective acylation is accomplished by adding an acylating agent, such as acetyl chloride, in the presence of a lithium base, preferably n-butyl lithium. The resulting baccatin III may be used in processes for forming paclitaxel.
U.S. Pat. No. 5,688,977 issued Nov. 18, 1997 to Sisti et al, WO 97/31911 and WO 97/34866 describe an efficient methodology to synthesize docetaxel (TAXOTERE(copyright)). These references teach a method for docetaxel synthesis comprising the esterification of C-7, C-10 di-CBZ 10-deacetylbaccatin III and an N-CBZ C-2-hydrogenatable benzyl-type protected 3-phenyl isoserine side chain. In that process, however, neither acylation of the C-10 hydroxyl nor benzolation of the C-3xe2x80x2 nitrogen was necessary.
Despite the advance made in the semi-synthesis of the paclitaxel molecule in the above described processes, there remains a need for more efficient protocols for the synthesis of paclitaxel in order to increase efficiencies in yields and production rates. There remains such a need for semi-synthesis that may be implemented into commercial processes. There is a further need for efficient protocols for the synthesis of paclitaxel analogs, intermediates and various A-ring side chain structures.
It is an object of the present invention to provide a new and useful method for producing paclitaxel or paclitaxel analogs.
A further object of the present invention is to provide a new, useful and efficient protocol for the attachment of a protected A-ring side chain to a protected baccatin III skeleton which may then be converted into paclitaxel or a paclitaxel analog.
It is another object of the present invention to provide a new and useful protocol for the semi-synthesis of paclitaxel and analogs thereof in an effort to provide a high yield of paclitaxel and paclitaxel analogs in a cost efficient manner.
Yet another object of the present invention is to provide a method for the production of paclitaxel and analogs thereof which potentially can be called to commercial implementation.
According to the present invention, then, a new and useful method of producing paclitaxel or a paclitaxel analog is provided. According to the general method, C-7, C-10 di-CBZ 10-deacetylbaccatin III of the formula 
is esterified with an N-carbamate protected, C-2-protected 3-phenyl isoserine side chain of the formula 
to form a first intermediate compound of the formula 
wherein P1 is a hydroxyl protecting group and R1 is chosen from the group consisting of Ph, PhCH2, an aromatic group, an alkyl group, and an olefinic group. Next, hydrogen is substituted for the C-7, C-10 carbobenzyloxy groups in the first intermediate compound and R2CO is substituted for the R1OCO group at the C-3xe2x80x2 nitrogen site to form a second intermediate compound of the formula 
wherein P1 is a hydroxyl protecting group and R2 is chosen from the group consisting of Ph, PhCH2, PhOxe2x80x94PhCH2Oxe2x80x94, an aromatic group, an alkyl group, an olefinic group, an O-aromatic group, an O-alkyl group, and an O-olefinic group. Thereafter, the second intermediate compound is acylated at the C-10 hydroxyl position to form a third intermediate compound of the formula 
wherein P1 and R2 are as above. Finally, the third intermediate compound is deprotected by substituting hydrogen for P1 to produce paclitaxel or a paclitaxel analog. It is preferred that P1 be a hydrogenatable benzyl-type protecting group, in particular benzyloxymethyl or benzyl, with benzyloxymethyl being the preferred protecting group. Also, it is preferred that R1 is PhCH2 and R2 is Ph, thereby to produce paclitaxel.
The esterification reaction is preferably performed by dissolving the isoserine side chain and the C-7, C-10 di-CBZ 10-deacetylbaccatin III in toluene to form a first solution after which dimethylamino pyridine (DMAP) and a dialkylcarbodiimide are added to the first solution to produce a second solution containing the first intermediate compound. The step of substituting hydrogen for the C-7 and C-10 carbobenzyloxy groups and substituting R2CO for the R1OCO group at the C-3xe2x80x2 nitrogen site may be conducted first to produce an amine or an amine salt of the formula 
wherein P1 is a hydroxyl protecting group and wherein R3 is selected from the group consisting of NH2 and NH3+Xxe2x88x92 wherein X is a deprotonated organic acid, preferably deprotonated trifluroacetic acid, after which R2CO is attached at the C-3xe2x80x2 nitrogen site to produce the second intermediate compound. The step of deprotecting the third intermediate compound may be accomplished by dissolving the third intermediate compound in isopropanol and hydrogenating in a presence of Pearlman""s catalyst.
The present invention is also directed at a method of acylating a 10-hydroxy paclitaxel analog for use in the production of paclitaxel and paclitaxel analogs. The method provides for selective acylation at the C-10 hydroxyl position over the C-7 hydroxyl position. According to the general method, a selected quantity of a 10-hydroxy paclitaxel analog of the formula: 
wherein P1 is a hydroxyl protecting group and R2 is chosen from the group consisting of Ph, PhCH2, PhOxe2x80x94PhCH2Oxe2x80x94, an aromatic group, an alkyl group, an olefinic group, an O-aromatic group, an O-alkyl group, and an O-olefinic group, is dissolved in an acceptable ether solvent therefor to form a first solution at a first temperature. The first solution is then cooled to a second temperature and at least one equivalent of an alkali base is added to the first solution at the second temperature to form a first intermediate in a second solution, said first intermediate having a formula: 
wherein M is an alkali metal, and P1 and R2 are as above. To the first intermediate in the second solution at the second temperature is then added at least one equivalent of an acylating agent to form a third solution, such that a compound of the formula 
wherein P1 and R2 are as above, is formed in the third solution. Preferably, M is selected from the group consisting of lithium, potassium and sodium, R2 is Ph and P1 is a hydrogenatable benzyl-type protecting group, in particular benzyloxymethyl or benzyl. Further preferred is where M is lithium and P1 is benzyloxymethyl.
The present invention is also directed to an alternative method of acylating a 10-hydroxy paclitaxel analog for use in the production of paclitaxel and paclitaxel analogs. According to the general method, a selected quantity of a 10-hydroxy paclitaxel analog of the formula: 
wherein P1 is a hydroxyl protecting group and R2 is chosen from the group consisting of Ph, PhCH2, PhOxe2x80x94PhCH2Oxe2x80x94, an aromatic group, an alkyl group, an olefinic group, an O-aromatic group, an O-alkyl group, and an O-olefinic group, is dissolved in an acceptable ether solvent therefor to form a first solution. Next, a solution containing an alkali salt is mixed into the first solution to form a second solution. Next, a base selected from a group consisting of trialkyl amine bases and pyridine is added to the second solution thereby to form a third solution. The third solution is then combined with an acylating agent, preferably acetyl chloride, to form a fourth solution such that a compound of the formula 
wherein P1 and R2 are as above, is formed in the fourth solution.
In this method, it is preferred that R2 is Ph and P1 is a hydrogenatable benzyl-type protecting group, in particular benzyloxymethyl or benzyl. The alkali salt may be selected from the group consisting of a lithium salt, a potassium salt and a sodium salt. The alkali salt is preferably a lithium salt, such as lithium chloride or lithium iodide.
The present invention is also directed to a chemical intermediate for use in producing paclitaxel or paclitaxel analogs, said intermediate having the formula: 
wherein M is an alkali metal, P1 is a hydroxyl protecting group and R2 is chosen from the group consisting of Ph, PhCH2, PhOxe2x80x94PhCH2Oxe2x80x94, an aromatic group, an alkyl group, an olefinic group, an O-aromatic group, an O-alkyl group, and an O-olefinic group. Preferably, P1 is a hydrogenatable benzyl-type protecting group, in particular benzyloxymethyl or benzyl, with benzyloxymethyl preferred. M may be selected from the group consisting of lithium, potassium and sodium. Preferably, M is lithium and R2 is Ph.
These and other objects of the present invention will become more readily appreciated and understood when the following detailed description of the exemplary embodiments is considered.
The present disclosure is broadly directed to a chemical process for the efficient production of paclitaxel and paclitaxel analogs as well as intermediates and precursors therefor. More specifically, the present invention is directed to a method of producing paclitaxel and paclitaxel analogs using a taxane backbone that is protected at the C-7 and C-10 positions with the carbobenzyloxy (CBZ) protecting,group.
The general process described herein involves the production of the C-7, C-10 di-CBZ 10-deacetylbaccatin III backbone, the production of the suitably protected 3-phenylisoserine acid having a hydroxyl protecting group at C-2, the condensation of the two compounds, and the subsequent deprotection at C-7 and C-10, as well as at the C-3xe2x80x2 nitrogen site as described in U.S. Pat. No. 5,688,977. Acylation at the C-3xe2x80x2 nitrogen site is performed followed by selective acylation at the C-10 hydroxyl site over the C-7 hydroxyl site to add the acetyl group followed by further deprotection to yield paclitaxel or a paclitaxel analog.